Volume 306, number 2,3, 181-184 FEBS 11301 © 1992 Federation of European BiochemicalSocieties 00145793/9~$5.00
July 1992
Molecular cloning of a novel ras-like protein from chicken J u d i t h T r u e b a n d Beat T r u e b Laboratorhon fflr Btochemie L Etdgent~ssische Technische Hochschule, CH-8092 Zflrich, Switzerland Received May 20 1992 We have isolated a eDNA clone from a chicken DNA expression library which code5 for a ras-lik¢ polypeptide of 216 amino acid n.~idues.This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil. two novel proteins that may be involvedin the coordination of the cell cycle. In the analno-terminal region, the three polypeptides possess a P-loop motif characteristic of GTP-binding proteins. At the carboxy.terminalend, however, they lack the typical CAAX-box which i~ usually responsible for membrane anchorase of ras-llke proteins. It is therefore likely that the three polypeptidesdefine a new subclass of GTP-binding proteins within the ras.like s.perfamily. Ras-like; Protooncogene;GTPase; GTP-binding; Spil; Chicken
1. I N T R O D U C T I O N Ras-like proteins constitute a superfamily o f low molecular weight proteins that share 30-55% sequence identity among each other [1-4]. So far, more than forty different members of this superfamily have been identified which can be grouped into five distinct subfamilies, the ras-, ral-, rap-, rho-and rab-proteins. All ras-like proteins bind G T P with high affinity and catalyze its hydrolysis to GDP. In this way, the proteins act as molecular switches which are biologically active in the GTP-bound form, but inactive in the G D P - b o u n d form. It is believed that ras proteins function as transducers of mitogenic signals from growth factor receptors to downstream targets. R h o proteins m a y play a role in controlling the architecture o f the cytoskeleton, whereas rab proteins appear to be involved in the regulation of the secretory pathway. All ras-like proteins exhibit a similar domain structure. The amino-terminal region (residues 1-165 in Hras) represents the catalytic domain which binds and hydrolyses GTP. The following 20 residues form a heterogeneous region that differs largely a m o n g distinct ras-like proteins. The four earboxy-terminal residues, called the CAAX-box (where C is cysteine, A any aliphatie amino acid and X any uncharged amino acid), are responsible for membrane attachment which is essential for biological activity. This attachment is accomplished by several post-translational modifications ineluding farnesylation and palmitylation ofcarboxy-terminal cysteine residues. g a s genes have attracted considerable attention over the past ten years because they acquire transforming Correspondence: B. Trueb, Bioehemie I, ETH.Zentrum, CH-8092 Ztirich, Switzerland. Fax (41) (1) 261 5677,
Published by Elsevier Science Publi#her~'B.V.
properties by point mutations that affect their GTPbinding region (primarily codons 12-13 and 59-61). In fact, activated ras oncogenes have been identified in many bladder carcinomas and in about 30% of all other human tumors. We have been interested for some time in collagenoas proteins of the extracellular matrix [5,6]. Screening of an expression library for such a protein fortuitously led to the isolation of a e D N A clone which encoded a novel ras-like protein. This protein revealed an extraordinary degree o f sequence conservation between man, chicken and yeast and might therefore play a crucial role in normal cell growth, 2. M A T E R I A L S A N D M E T H O D S A 2gtl I expression library was screen~ with antibodies against type VI collagenas previouslydescribed [5,6l. lmmunorcactiv¢clones were amplified, subcloned into the sequencing v~etors Ml3mpl8 or M 13mp19 and sequenced on both strands by the dideoxychain termination method [7]. The sequences were analyzed with the software computer package of tile Oeneties Compute~ Group (University of Wisconsin, Madison WI). Total RNA was prepared from 15-day-old chicken embryos and from several cell lines [8] by the guanidinium-isothiocyanatemethod [9]. For Northern blots, 15/q~of total RNA was resolvedon a fm'maldehyde containing i% agaro~¢ gel and transferred to nitro¢¢llalo~ [10]. The nitrocellulosesheet was hybridizedunder standard conditions to the eDNA probe which had b~n label~l with [g-s~P]dCTPby the random primed oligolabelingmethod [11]. 3. R E S U L T S A N D DISCUSSION 3.1. Isolation and characterization o f eDNA clone Ju93 Screening o f a e D N A expression library with antibodies for a eollagenous glycoprotein furtuitously led to the isolation o f e D N A clone Ju93. Sequencing studies indicated that this clone did not code for a eollagenous
181
Volume 306, number 2,3 1 1
FEBS LETTERS
GAATTCCGGGAJ~AGACCCAGCATGGCCGCCCAAGGAGAGCCCCAAGTGCAGTTTAAGCTT M A A Q G ~ P C V Q Y M L
60 13
GTCCTGGTTG G CG A T G G T G G C A C T G G T A ~ C A A C A T T T G T A A A A C G T C A T ~ V L V G D G G T G K T T P V M R M L
ACTGGT T G
1.20 33
121 34
G AATTTG~%GAA G/%AATACGTA~CAACATTGG GTGTTGAAGTTC ATCCACTGGTATTCCAT E ~ E R M ~ V A T L G V E V H P L V ~ 14
180 53
I~I 5~
ACTAATfiGAG GCC CTATTAAATTTAATGTAT G G GAC/%~AG CTGGCCAG G A G A A G T T T G G T T |1 II G P I ~ ~ ~ V W D T A G Q ~ ~ F G
2 ti~ 73
241 74
G GTCTG CGAGATG GCTATTACATCC AAGCTC A G T G T G C C A T T A T A A T G T T T G A T G T A A C A G L ~ D G %' • I Q A 0 C A ~ I M F D V T
300 93
301 94
TCAJ%GAGTTACTTACAAGAATGTAC CTAACTGGCATAG~G AC CTGGTACGGGTATGTG KA S ~ V T ¥ R N V P H W II R D L V R V C E
360 I13
36~ 114
AACAVC CCTATAGTfiTTGTGTGGCAACAAAGTGGATA ,"~A~ ~ G~,CAG ;~AI~GTC AAG GCA N I ~ I V L C G N K V D I K D R ~( V K A
420 133
421 134
AAATCCAT'2GTCTTCCACAGGAAQAJ%OAATC TCCAOThTTAT GACAT£ TC AGC C AAGAGT K S I V ~ H R ~ K H L Q ~ y D 1 S A M S
480 153
481 154
A A C T A C A A C T T T G AGAAGC CGTTCCTCTGGCTTGCTAGGAAGCTAATTG GAGATCCTAAC H Y B F E K P F L H L A R R L I G D P .~1
540 193
541 ~74
TTGGAA'/TTGTTGCCATGCCTGCTCTT~CACCACCTGAAGTT~ GTTATGGACCC~GCACTG L Z P V A M P /% L A P P E V V M D P A L
600 193
501 194
GCAGC ACAGTATGAGCAAGACTTAC AGATTGCTCAAACCAC~GCACTG CC AGATGA~SG~T A A Q Y E Q D L Q : A Q T T ~% L P D E D
660 213
561 21,1
GATGACCTGTGAGGGATGAAGCTGGAGCCCAGCG~CAGAAGTCTAG~TTTATA~GCAACT D D t. •
"/20 216
?21
GTCCTG?GATG?CAGTGGTGCAGCGTGTTTGCCACT ."~ATTATCTAGCTGAGCAGAACA~
780
981
GTGCTTAATCTTTGGGATGCTGAAAGAGATGAATGGGCTTCGCAGTGAATGTGGCAGTTC
840
61 14
July 1992
kb
841
A p J ~ A C G A A J ~ C A A C A P J i A C .'~TCATAATTTTGGACC T C C A T A T ~ A G C TGTT TTTTG G
900
901
ACTGCP~TTACTTCCCCTTTGAGTTTC~TATAA~AeTGCTGCAGTCACATCAAAGTGT
960
961
TATGTGGTAJ%ATCTTGTTT CTGTCA .~TCCCGGAATTC
2.37
1.35
1
2
3
Fig. 2. Northern blot analysis of eDNA clone Ju93 with total RNA from chicken embryos(lane 1),chicken fibroblasts(lane 2) and human VAI3 cells (lane 3). The milFation positions of standard RNA molecules are indicated in the right margin.
997
On Northern blots with R N A preparations from chicken embryos and chicken fibroblasts, our e D N A clone detected a major band of 1,300 nucleotides and a minor band of 2,100 nueleotides (Fig. 2). Similar bands were obtained with R N A preparations from human VA 13 cells, suggesting that both, avian and mammalian cells express the 3u93 gene. 3.2. Similarity of Ju93 to ras-like proteins A detailed comparison of the Ju93 polypeptide sequence with all entries of the Swissprot databank revealed substantial similarities with members of the raglike superfamily (Fig. 3). With H-ras, N-ras, ral-A, rapG1
H-Eas
4
-4, 0 . 2 4
polylmptide (Fig. 1). The nucleotide sequence contained an A T G codon (positions 22-24) in a surroundin~ that was consistent with a typical start site of transiation [12]. This initiation codon was followed by an open reading frame of 648 bp. The amino acid sequence derived from the nucleotide sequence predicted a protein o f 216 residues with a molecular mass of 24,427 Da. At position 17-24, this sequence contained a P-loop motif ( G D G G T G K T ) which is characteristic of GTP-binding proteins [13]. 1
4,40
•. ~
Fig. 1. Nu¢leotldeand derived amino acid sequence of eDNA clone Ju93.
Ju93
•- ,
G2
MAAQGEP QVQFKLVL~TrV~RH • ::L
1
I I :Ill,
I l.I
LTG ~ ' ~ V - ~ I1..:,
.
:
..I
.41
LGV~V~L :11: :
•
".
MTZYKLVVV~I%G GVG K ~ A L T I QL Z Q I ~ M F V D ~ D P~I ~D S YRKQ
50 43
G3 Ju93
51
VFHTNRGPIKFNVW~-~KFGGLRDGYYIQAQCA~IMFDVTSRV'2YKN
H-=as
44
vv ZD G R~'CL. LD l L ~ . T . ~ . ~ I I S A M R D Q I M R T O E
J~93
101
I . . . . .
93
illillllllll,
::
i:.':ll.l
G4 ,C ~.N ZP £ V L C ~ ' ~ Z
V~WaRDLVRV, :,,i:
l~-=as
:
11
ii,
:,:1.:.,
KD RMVZ~AK •S T VF IiR~.XN L~ YY
::iilill.llll.li'..I.l.,:
.
I00
,i.:
GI LCVP AT NNTK SFED
:
1,
92
X49
.:.1.
ZIIQ~RZQ II~RVKDSDDVPMVLV~NI~eDILP A R T V Z T B Q A Q D L A R S Y G
ZP~I
142
G5
Ju93
148
~NYNPERP l:,llll
....
PLWLARKLZ GDPNLEFVAMPALAPPEVVMD~ALAAQY I.:1,
l.l,:
,.,
Ju%3
198
~QDLQ£AQTTALPD~DDDL
21~
H-EaS
187
VZS
189
:.
I.I1:
197
...:,,.:
Fig. 3. Alignment of the amino acid sequence of the chicken proteins Ju93 and H-rag. The five conserved regions involved in G T P metabolism are boxed.
182
Vo!ume 306, number 2,3
July 1992
FEBS LETTERS
YEAST HUMAN CHICK
1 1 1
MA-QPQNVPTFRLVLVGDGGTGKTTPVKRHLTGE?ERRYIATLGVEVHPLHFHTNFGE~C MAAQGEPQVQFKLVLVGDGGTGKTT~VKRHLTGEFEMRYVATLGVEVHPLVFMTNHGPIK MAAQGEgOVQFKLVLVGDGGTGRTTFVKRNLTGEFEKKYVATLGVSVHPLV~HTNRG~IK
5~ 60 60
YEAST HUMAN CHICK
60
~i 61
FNVNDTAGQEKLGGLRDGYYIQGQCGIXMFDVTSRITYRNVPHW~RDLVRVCENI~IvLe ~NVWDTAGQSKFGGLBDGMYIQAQCA~IMFDVTSRVTYKNV~WHRDLVRVCENIPIVLC FNVWDTAGQEKFGGLRDGYY~QAQCAI~H~DVTSBVTYKNVPNWHRDLVRVCEHIPIVLC
119 1~0 i~0
HUMAN CHICK
120 12% 121
GNRVDVEEBKVRAKAITFHRRKNL~YYDISARSNYNFERPFLWLAR~LVGN~NLEFVASP GNKV01KDRKVKAKSZV~NBKKHLQY~DISARSNYNFERPFLWLARKL~GD~NLEPVAMP GNKVDIKDR~VRAKSIVFHRRKNLQYYDISAMSNYNFEKPFLWLABKLIGDPNLE~VAMP
179 IS0 180
MZAST HUMAN CHICK
180 181 181
ALAPPEVQVDQQLLAQYQO~MN~AAAMPLPDEDDADL ALAPPEVVMDPALAAQY~HDLEVAQTTALPDEDD-DL ALAPPEVVMDPALAAQY~QDLQIAQTTALPD~DD-DL
216 216 216
Fig. 4. Alignm,~nt of th~ amino acid sequen~s of the chicken protein Ju93, the human protein TC4 and 1he yeast protein Spil. Identical positions are marked by asterisks, conservative replacements by dots.
I B, tab, Rho-A and YPT1, the Ju93 protein shared 25-30% sequence identity. If con-:ervative amino acid substitutions were included, these similarities increased to 49-53%. As is the case for most ras-like proteins [2], the Ju93 sequence contained five particularly well conserved regions (G-1 to G 5 ) which are likely to be involved in GTP metabolism (Fig. 3). In contrast to other ras-like proteins, however, the typical CAAX-box was missing at the carboxy-terminus of the polypeptide sequence. Since this sequence motif is responsible for membrane attachment, the Ju93 protein might exist in a soluble, not membrane-anchored form. 3.3. Ju93, TC4 and Spil define a new subclass tv#hin the ras-like superfamily A strikingly high homology was observed between the Ju93 sequence and two recent databank entries, human TC4 [14] and yeast Spil [15]. At the nucleotide level, the chicken sequence shared 68% identity with the yeast sequence and 89% identity with the human sequence. Under the assumption that the human sequence contained a minor sequencing error at codon 179o all three eDNA sequences could be translated into polypeptides of 216 amino acid residues. Compared to the (corrected) human amino acid sequence, the chicken polypeptide showed three amino acid substitutions (Fig. 4). All these substitutions occurred within the carboxyterminal domain corresponding to the heterogeneous region of ras-like proteins [1--4]. Compared to the yeast protein, the chicken polypeptide displayed 82% sequence identity or 89% similarity, if conservative amino acid replacements were included. Since all three proteins lacked the characteristic CAAX box at their carboxy termini, they may define a new subfamily of GTPase signaling proteins within the ras-like superfamily. The extraordinary degree of conservation throughout metazoan evolution strongly implies that the three novel
proteins serve a crucial function in normal cell growth. What this function is, can only be speculated at present. Little is known about TC4 except that it was isolated from a teratocareinoma eDNA library with a mixed oligonucleotide probe specific for ras-like sequences [14]. More information, however, is available on Spil from fission yeast [15]. Overexpression of Spil was found to resctte piml cells which undergo premature initiation o f mitosis before completion of DNA replication. Thus, Spil appears to be involved in the maintenance of a coordinated cell eyele. The loss o f a single copy of the Spil gene caused a dramatic instability of the genetic material and eventually led to mitotic haploidization. Since a wide variety of tumor cells display an abnormal karyotype, it seems conceivable that the functional loss of the Spil gene may contribute to the malignant transformation of a a l l by affecting its genetic stability. It would therefore be interesting to examine the effects of overexpressing the Ju93 gene in normal cells or of introducing small mutations into the Ju93 sequence which may affect its GTP metabolism. Acknowledgements: This work was supported by Grants 31-30881.91 and 31-9065.87 from the Swiss National Science Foundation and by a grant from the ETH Ztirich.
REFERENCES [1] Barbacid, M. (1987) Annu. Rev. Bicthem. 56, 779-827. [2] Bourne. H.R., Sanders. D.A. and McCormick, F. (1991) Nature 349. 117-127. [31 Wittinghofer, A. and Pal, E.F. (1991) Trends Bioehem. Sei. 16. 382-387. [4] Downward, J. (1990) Trends Biochem. ~:i. 15, 469--472. [5] Trueb, B.. Sehaeren.Wiemers. N., Sehreier, T. and Winterhalter, K,H, (1989) J. Biol. Chem. 264, 136-140, [61 Koller, E.. Winterhalter. K.H. and Trueb, B. (1989) EMBO J. 8. 1073-1077. [7] Sanger, F., Nickkn, S. and Coulson, A.R.. (1977) Proe. Natl. Aead. Sci. USA 74, 5463-5467.
183
Volume 306, number 2,3
FEBS L E T T E R S
[8] Schreier, T., Frils, R.R., Winterhalter, K,H. and Trueb, B. (1988) Biochem, J, 253, 381-386. [9] Chomczynski, P. and Sa¢chi, N. (1987) Anal, Bioehem, 162, 156159. [10] Maniatis, T,, Fritsch, E,F. and Sambrook, J, (1982) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
184
July 1992
[1I] Feinberg, A. and Vogelstein, B. (1983) Anal, Biochem, ! 32, 6-13. [12] Ko:'ak, M, (1987) Nucleic Acids Res, 15, 8125-8148, [13] Saraste, M., Sibbald, P,R. and Wittinghofer, A. (1990) Trends Biochem. Sci. 15, 430--434. [14] Drlvas, G.T.. Shlh, A., Coutavas. E,, Rush. M.G. and D'Eustaehio, P. (1990) Mol. Cell. Biol. 10, 1793-179~,. [15] Matsumoto, T. and Beach, D. (1991) Cell 66. 347-360.
July 1992
Molecular cloning of a novel ras-like protein from chicken J u d i t h T r u e b a n d Beat T r u e b Laboratorhon fflr Btochemie L Etdgent~ssische Technische Hochschule, CH-8092 Zflrich, Switzerland Received May 20 1992 We have isolated a eDNA clone from a chicken DNA expression library which code5 for a ras-lik¢ polypeptide of 216 amino acid n.~idues.This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil. two novel proteins that may be involvedin the coordination of the cell cycle. In the analno-terminal region, the three polypeptides possess a P-loop motif characteristic of GTP-binding proteins. At the carboxy.terminalend, however, they lack the typical CAAX-box which i~ usually responsible for membrane anchorase of ras-llke proteins. It is therefore likely that the three polypeptidesdefine a new subclass of GTP-binding proteins within the ras.like s.perfamily. Ras-like; Protooncogene;GTPase; GTP-binding; Spil; Chicken
1. I N T R O D U C T I O N Ras-like proteins constitute a superfamily o f low molecular weight proteins that share 30-55% sequence identity among each other [1-4]. So far, more than forty different members of this superfamily have been identified which can be grouped into five distinct subfamilies, the ras-, ral-, rap-, rho-and rab-proteins. All ras-like proteins bind G T P with high affinity and catalyze its hydrolysis to GDP. In this way, the proteins act as molecular switches which are biologically active in the GTP-bound form, but inactive in the G D P - b o u n d form. It is believed that ras proteins function as transducers of mitogenic signals from growth factor receptors to downstream targets. R h o proteins m a y play a role in controlling the architecture o f the cytoskeleton, whereas rab proteins appear to be involved in the regulation of the secretory pathway. All ras-like proteins exhibit a similar domain structure. The amino-terminal region (residues 1-165 in Hras) represents the catalytic domain which binds and hydrolyses GTP. The following 20 residues form a heterogeneous region that differs largely a m o n g distinct ras-like proteins. The four earboxy-terminal residues, called the CAAX-box (where C is cysteine, A any aliphatie amino acid and X any uncharged amino acid), are responsible for membrane attachment which is essential for biological activity. This attachment is accomplished by several post-translational modifications ineluding farnesylation and palmitylation ofcarboxy-terminal cysteine residues. g a s genes have attracted considerable attention over the past ten years because they acquire transforming Correspondence: B. Trueb, Bioehemie I, ETH.Zentrum, CH-8092 Ztirich, Switzerland. Fax (41) (1) 261 5677,
Published by Elsevier Science Publi#her~'B.V.
properties by point mutations that affect their GTPbinding region (primarily codons 12-13 and 59-61). In fact, activated ras oncogenes have been identified in many bladder carcinomas and in about 30% of all other human tumors. We have been interested for some time in collagenoas proteins of the extracellular matrix [5,6]. Screening of an expression library for such a protein fortuitously led to the isolation of a e D N A clone which encoded a novel ras-like protein. This protein revealed an extraordinary degree o f sequence conservation between man, chicken and yeast and might therefore play a crucial role in normal cell growth, 2. M A T E R I A L S A N D M E T H O D S A 2gtl I expression library was screen~ with antibodies against type VI collagenas previouslydescribed [5,6l. lmmunorcactiv¢clones were amplified, subcloned into the sequencing v~etors Ml3mpl8 or M 13mp19 and sequenced on both strands by the dideoxychain termination method [7]. The sequences were analyzed with the software computer package of tile Oeneties Compute~ Group (University of Wisconsin, Madison WI). Total RNA was prepared from 15-day-old chicken embryos and from several cell lines [8] by the guanidinium-isothiocyanatemethod [9]. For Northern blots, 15/q~of total RNA was resolvedon a fm'maldehyde containing i% agaro~¢ gel and transferred to nitro¢¢llalo~ [10]. The nitrocellulosesheet was hybridizedunder standard conditions to the eDNA probe which had b~n label~l with [g-s~P]dCTPby the random primed oligolabelingmethod [11]. 3. R E S U L T S A N D DISCUSSION 3.1. Isolation and characterization o f eDNA clone Ju93 Screening o f a e D N A expression library with antibodies for a eollagenous glycoprotein furtuitously led to the isolation o f e D N A clone Ju93. Sequencing studies indicated that this clone did not code for a eollagenous
181
Volume 306, number 2,3 1 1
FEBS LETTERS
GAATTCCGGGAJ~AGACCCAGCATGGCCGCCCAAGGAGAGCCCCAAGTGCAGTTTAAGCTT M A A Q G ~ P C V Q Y M L
60 13
GTCCTGGTTG G CG A T G G T G G C A C T G G T A ~ C A A C A T T T G T A A A A C G T C A T ~ V L V G D G G T G K T T P V M R M L
ACTGGT T G
1.20 33
121 34
G AATTTG~%GAA G/%AATACGTA~CAACATTGG GTGTTGAAGTTC ATCCACTGGTATTCCAT E ~ E R M ~ V A T L G V E V H P L V ~ 14
180 53
I~I 5~
ACTAATfiGAG GCC CTATTAAATTTAATGTAT G G GAC/%~AG CTGGCCAG G A G A A G T T T G G T T |1 II G P I ~ ~ ~ V W D T A G Q ~ ~ F G
2 ti~ 73
241 74
G GTCTG CGAGATG GCTATTACATCC AAGCTC A G T G T G C C A T T A T A A T G T T T G A T G T A A C A G L ~ D G %' • I Q A 0 C A ~ I M F D V T
300 93
301 94
TCAJ%GAGTTACTTACAAGAATGTAC CTAACTGGCATAG~G AC CTGGTACGGGTATGTG KA S ~ V T ¥ R N V P H W II R D L V R V C E
360 I13
36~ 114
AACAVC CCTATAGTfiTTGTGTGGCAACAAAGTGGATA ,"~A~ ~ G~,CAG ;~AI~GTC AAG GCA N I ~ I V L C G N K V D I K D R ~( V K A
420 133
421 134
AAATCCAT'2GTCTTCCACAGGAAQAJ%OAATC TCCAOThTTAT GACAT£ TC AGC C AAGAGT K S I V ~ H R ~ K H L Q ~ y D 1 S A M S
480 153
481 154
A A C T A C A A C T T T G AGAAGC CGTTCCTCTGGCTTGCTAGGAAGCTAATTG GAGATCCTAAC H Y B F E K P F L H L A R R L I G D P .~1
540 193
541 ~74
TTGGAA'/TTGTTGCCATGCCTGCTCTT~CACCACCTGAAGTT~ GTTATGGACCC~GCACTG L Z P V A M P /% L A P P E V V M D P A L
600 193
501 194
GCAGC ACAGTATGAGCAAGACTTAC AGATTGCTCAAACCAC~GCACTG CC AGATGA~SG~T A A Q Y E Q D L Q : A Q T T ~% L P D E D
660 213
561 21,1
GATGACCTGTGAGGGATGAAGCTGGAGCCCAGCG~CAGAAGTCTAG~TTTATA~GCAACT D D t. •
"/20 216
?21
GTCCTG?GATG?CAGTGGTGCAGCGTGTTTGCCACT ."~ATTATCTAGCTGAGCAGAACA~
780
981
GTGCTTAATCTTTGGGATGCTGAAAGAGATGAATGGGCTTCGCAGTGAATGTGGCAGTTC
840
61 14
July 1992
kb
841
A p J ~ A C G A A J ~ C A A C A P J i A C .'~TCATAATTTTGGACC T C C A T A T ~ A G C TGTT TTTTG G
900
901
ACTGCP~TTACTTCCCCTTTGAGTTTC~TATAA~AeTGCTGCAGTCACATCAAAGTGT
960
961
TATGTGGTAJ%ATCTTGTTT CTGTCA .~TCCCGGAATTC
2.37
1.35
1
2
3
Fig. 2. Northern blot analysis of eDNA clone Ju93 with total RNA from chicken embryos(lane 1),chicken fibroblasts(lane 2) and human VAI3 cells (lane 3). The milFation positions of standard RNA molecules are indicated in the right margin.
997
On Northern blots with R N A preparations from chicken embryos and chicken fibroblasts, our e D N A clone detected a major band of 1,300 nucleotides and a minor band of 2,100 nueleotides (Fig. 2). Similar bands were obtained with R N A preparations from human VA 13 cells, suggesting that both, avian and mammalian cells express the 3u93 gene. 3.2. Similarity of Ju93 to ras-like proteins A detailed comparison of the Ju93 polypeptide sequence with all entries of the Swissprot databank revealed substantial similarities with members of the raglike superfamily (Fig. 3). With H-ras, N-ras, ral-A, rapG1
H-Eas
4
-4, 0 . 2 4
polylmptide (Fig. 1). The nucleotide sequence contained an A T G codon (positions 22-24) in a surroundin~ that was consistent with a typical start site of transiation [12]. This initiation codon was followed by an open reading frame of 648 bp. The amino acid sequence derived from the nucleotide sequence predicted a protein o f 216 residues with a molecular mass of 24,427 Da. At position 17-24, this sequence contained a P-loop motif ( G D G G T G K T ) which is characteristic of GTP-binding proteins [13]. 1
4,40
•. ~
Fig. 1. Nu¢leotldeand derived amino acid sequence of eDNA clone Ju93.
Ju93
•- ,
G2
MAAQGEP QVQFKLVL~TrV~RH • ::L
1
I I :Ill,
I l.I
LTG ~ ' ~ V - ~ I1..:,
.
:
..I
.41
LGV~V~L :11: :
•
".
MTZYKLVVV~I%G GVG K ~ A L T I QL Z Q I ~ M F V D ~ D P~I ~D S YRKQ
50 43
G3 Ju93
51
VFHTNRGPIKFNVW~-~KFGGLRDGYYIQAQCA~IMFDVTSRV'2YKN
H-=as
44
vv ZD G R~'CL. LD l L ~ . T . ~ . ~ I I S A M R D Q I M R T O E
J~93
101
I . . . . .
93
illillllllll,
::
i:.':ll.l
G4 ,C ~.N ZP £ V L C ~ ' ~ Z
V~WaRDLVRV, :,,i:
l~-=as
:
11
ii,
:,:1.:.,
KD RMVZ~AK •S T VF IiR~.XN L~ YY
::iilill.llll.li'..I.l.,:
.
I00
,i.:
GI LCVP AT NNTK SFED
:
1,
92
X49
.:.1.
ZIIQ~RZQ II~RVKDSDDVPMVLV~NI~eDILP A R T V Z T B Q A Q D L A R S Y G
ZP~I
142
G5
Ju93
148
~NYNPERP l:,llll
....
PLWLARKLZ GDPNLEFVAMPALAPPEVVMD~ALAAQY I.:1,
l.l,:
,.,
Ju%3
198
~QDLQ£AQTTALPD~DDDL
21~
H-EaS
187
VZS
189
:.
I.I1:
197
...:,,.:
Fig. 3. Alignment of the amino acid sequence of the chicken proteins Ju93 and H-rag. The five conserved regions involved in G T P metabolism are boxed.
182
Vo!ume 306, number 2,3
July 1992
FEBS LETTERS
YEAST HUMAN CHICK
1 1 1
MA-QPQNVPTFRLVLVGDGGTGKTTPVKRHLTGE?ERRYIATLGVEVHPLHFHTNFGE~C MAAQGEPQVQFKLVLVGDGGTGKTT~VKRHLTGEFEMRYVATLGVEVHPLVFMTNHGPIK MAAQGEgOVQFKLVLVGDGGTGRTTFVKRNLTGEFEKKYVATLGVSVHPLV~HTNRG~IK
5~ 60 60
YEAST HUMAN CHICK
60
~i 61
FNVNDTAGQEKLGGLRDGYYIQGQCGIXMFDVTSRITYRNVPHW~RDLVRVCENI~IvLe ~NVWDTAGQSKFGGLBDGMYIQAQCA~IMFDVTSRVTYKNV~WHRDLVRVCENIPIVLC FNVWDTAGQEKFGGLRDGYY~QAQCAI~H~DVTSBVTYKNVPNWHRDLVRVCEHIPIVLC
119 1~0 i~0
HUMAN CHICK
120 12% 121
GNRVDVEEBKVRAKAITFHRRKNL~YYDISARSNYNFERPFLWLAR~LVGN~NLEFVASP GNKV01KDRKVKAKSZV~NBKKHLQY~DISARSNYNFERPFLWLARKL~GD~NLEPVAMP GNKVDIKDR~VRAKSIVFHRRKNLQYYDISAMSNYNFEKPFLWLABKLIGDPNLE~VAMP
179 IS0 180
MZAST HUMAN CHICK
180 181 181
ALAPPEVQVDQQLLAQYQO~MN~AAAMPLPDEDDADL ALAPPEVVMDPALAAQY~HDLEVAQTTALPDEDD-DL ALAPPEVVMDPALAAQY~QDLQIAQTTALPD~DD-DL
216 216 216
Fig. 4. Alignm,~nt of th~ amino acid sequen~s of the chicken protein Ju93, the human protein TC4 and 1he yeast protein Spil. Identical positions are marked by asterisks, conservative replacements by dots.
I B, tab, Rho-A and YPT1, the Ju93 protein shared 25-30% sequence identity. If con-:ervative amino acid substitutions were included, these similarities increased to 49-53%. As is the case for most ras-like proteins [2], the Ju93 sequence contained five particularly well conserved regions (G-1 to G 5 ) which are likely to be involved in GTP metabolism (Fig. 3). In contrast to other ras-like proteins, however, the typical CAAX-box was missing at the carboxy-terminus of the polypeptide sequence. Since this sequence motif is responsible for membrane attachment, the Ju93 protein might exist in a soluble, not membrane-anchored form. 3.3. Ju93, TC4 and Spil define a new subclass tv#hin the ras-like superfamily A strikingly high homology was observed between the Ju93 sequence and two recent databank entries, human TC4 [14] and yeast Spil [15]. At the nucleotide level, the chicken sequence shared 68% identity with the yeast sequence and 89% identity with the human sequence. Under the assumption that the human sequence contained a minor sequencing error at codon 179o all three eDNA sequences could be translated into polypeptides of 216 amino acid residues. Compared to the (corrected) human amino acid sequence, the chicken polypeptide showed three amino acid substitutions (Fig. 4). All these substitutions occurred within the carboxyterminal domain corresponding to the heterogeneous region of ras-like proteins [1--4]. Compared to the yeast protein, the chicken polypeptide displayed 82% sequence identity or 89% similarity, if conservative amino acid replacements were included. Since all three proteins lacked the characteristic CAAX box at their carboxy termini, they may define a new subfamily of GTPase signaling proteins within the ras-like superfamily. The extraordinary degree of conservation throughout metazoan evolution strongly implies that the three novel
proteins serve a crucial function in normal cell growth. What this function is, can only be speculated at present. Little is known about TC4 except that it was isolated from a teratocareinoma eDNA library with a mixed oligonucleotide probe specific for ras-like sequences [14]. More information, however, is available on Spil from fission yeast [15]. Overexpression of Spil was found to resctte piml cells which undergo premature initiation o f mitosis before completion of DNA replication. Thus, Spil appears to be involved in the maintenance of a coordinated cell eyele. The loss o f a single copy of the Spil gene caused a dramatic instability of the genetic material and eventually led to mitotic haploidization. Since a wide variety of tumor cells display an abnormal karyotype, it seems conceivable that the functional loss of the Spil gene may contribute to the malignant transformation of a a l l by affecting its genetic stability. It would therefore be interesting to examine the effects of overexpressing the Ju93 gene in normal cells or of introducing small mutations into the Ju93 sequence which may affect its GTP metabolism. Acknowledgements: This work was supported by Grants 31-30881.91 and 31-9065.87 from the Swiss National Science Foundation and by a grant from the ETH Ztirich.
REFERENCES [1] Barbacid, M. (1987) Annu. Rev. Bicthem. 56, 779-827. [2] Bourne. H.R., Sanders. D.A. and McCormick, F. (1991) Nature 349. 117-127. [31 Wittinghofer, A. and Pal, E.F. (1991) Trends Bioehem. Sei. 16. 382-387. [4] Downward, J. (1990) Trends Biochem. ~:i. 15, 469--472. [5] Trueb, B.. Sehaeren.Wiemers. N., Sehreier, T. and Winterhalter, K,H, (1989) J. Biol. Chem. 264, 136-140, [61 Koller, E.. Winterhalter. K.H. and Trueb, B. (1989) EMBO J. 8. 1073-1077. [7] Sanger, F., Nickkn, S. and Coulson, A.R.. (1977) Proe. Natl. Aead. Sci. USA 74, 5463-5467.
183
Volume 306, number 2,3
FEBS L E T T E R S
[8] Schreier, T., Frils, R.R., Winterhalter, K,H. and Trueb, B. (1988) Biochem, J, 253, 381-386. [9] Chomczynski, P. and Sa¢chi, N. (1987) Anal, Bioehem, 162, 156159. [10] Maniatis, T,, Fritsch, E,F. and Sambrook, J, (1982) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
184
July 1992
[1I] Feinberg, A. and Vogelstein, B. (1983) Anal, Biochem, ! 32, 6-13. [12] Ko:'ak, M, (1987) Nucleic Acids Res, 15, 8125-8148, [13] Saraste, M., Sibbald, P,R. and Wittinghofer, A. (1990) Trends Biochem. Sci. 15, 430--434. [14] Drlvas, G.T.. Shlh, A., Coutavas. E,, Rush. M.G. and D'Eustaehio, P. (1990) Mol. Cell. Biol. 10, 1793-179~,. [15] Matsumoto, T. and Beach, D. (1991) Cell 66. 347-360.
